Mar. Drugs 2013, 11, 20-32; doi:10.3390/md11010020
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Nutritional and Chemical Composition and Antiviral Activity of
Cultivated Seaweed Sargassum naozhouense Tseng et Lu
Yan Peng 1, Enyi Xie 2, Kai Zheng 3, Mangaladoss Fredimoses 1, Xianwen Yang 1, Xuefeng Zhou 1,
Yifei Wang 3, Bin Yang 1, Xiuping Lin 1, Juan Liu 1 and Yonghong Liu 1,*
1
2
3
Key Laboratory of Marine Bio-resources Sustainable Utilization, Guangdong Key Laboratory of
Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of
Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;
E-Mails: [email protected] (Y.P.); [email protected] (M.F.); [email protected] (X.Y.);
[email protected] (X.Z.); [email protected] (B.Y.); [email protected] (X.L.);
[email protected] (J.L.)
Fisheries College, Guangdong Ocean University, Zhanjiang 524025, China;
E-Mail: [email protected]
Guangzhou Jinan Biomedicine Research and Development Center, Jinan University,
Guangzhou 510632, China; E-Mails: [email protected] (K.Z.); [email protected] (Y.W.)
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel./Fax: +86-20-8902-3244.
Received: 29 October 2012; in revised form: 29 November 2012 / Accepted: 5 December 2012 /
Published: 27 December 2012
Abstract: Sargassum naozhouense is a brown seaweed used in folk medicine and applied
for thousands of years in Zhanjiang, Guangdong province, China. This study is the first
time to investigate its chemical composition and antiviral activity. On the dry weight basis,
this seaweed was constituted of ca. 35.18% ash, 11.20% protein, 1.06% lipid and 47.73%
total carbohydrate, and the main carbohydrate was water-soluble polysaccharide. The
protein analysis indicated the presence of essential amino acids, which accounted for
36.35% of the protein. The most abundant fatty acids were C14:0, C16:0, C18:1 and
C20:4. The ash fraction analysis indicated that essential minerals and trace elements, such
as Fe, Zn and Cu, were present in the seaweed. IR analysis revealed that polysaccharides
from cultivated S. naozhouense may be alginates and fucoidan. The polysaccharides
possessed strong antiviral activity against HSV-1 in vitro with EC50 of 8.92 μg/mL. These
results demonstrated cultivated S. naozhouense has a potential for its use in functional
foods and antiviral new drugs.
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Keywords: Sargassum naozhouense; seaweed; chemical composition; antiviral activity
1. Introduction
Seaweeds, classified into red algae (Rhodophyta), brown algae (Ochrophyta, Phaeophyceae) and
green algae (Chlorophyta) [1,2], are a renewable natural resource with extensive distribution along the
Pacific coast [3]. They have been used mainly for human consumption (e.g., as food or as crude drugs
to treat gallstone, stomach ailment, eczema, cancer, renal disorders, scabies, psoriasis, asthma,
arteriosclerosis, heart disease, lung diseases and ulcers) and extraction of hydrocolloids, such as agar,
carrageens and alginates, but are still under-exploited [3–10]. In recent years, seaweeds have caused
emerging interest in biomedicine and the food area, because they possess a wealth of bioactive
compounds (such as sulfated polysaccharides, carotenoids, dietary fiber, protein, essential fatty acids,
vitamins, minerals, terpenoids, oxylipins, phlorotannins and steroids) with potential industrial and
agricultural applications [11–16]. For example, alginates from brown algae are often used as additives to
ameliorate the texture of food [7]. Therefore, seaweeds are a promising renewable resource with
considerable commercial potential for further exploitation.
Brown seaweeds (e.g., Sargassum fusiforme and Saccharina japonica—formerly Laminaria japonica)
have been used as Traditional Chinese Medicines in China for thousands of years [17,18].
Sargassum naozhouense Tseng et Lu, an edible brown algae widely distributed along the coasts of
Zhanjiang, Guangdong province, China, is commonly consumed as a sea vegetable or as crude drugs for
treating internal heat, infections, laryngitis and other ailments in locals [19]. According to literature
reports, the wild S. naozhouense is rich in polysaccharides, amino acids and trace elements [19].
However, little is known about the cultivated S. naozhouense, especially its nutritional and functional
properties. For full utilization of this rich resource, it is imperative to evaluate the nutritional and
functional properties of the cultivated S. naozhouense. In general, the nutritional properties are usually
estimated by the chemical composition. Moreover water-soluble sulfated polysaccharides are the main
constituents of seaweed cell walls, with potent antiviral activities, particularly against HSV [20,21].
Therefore, the aim of this study was to investigate the chemical composition of cultivated
S. naozhouense and the anti-HSV activity of water-soluble polysaccharide from cultivated S. naozhouense.
2. Results and Discussion
2.1. Chemical Composition of Cultivated S. naozhouense
The ash, protein and total carbohydrate were the most abundant constituents in S. naozhouense
(Table 1). The average contents of protein and ash were 11.20% and 35.18% in dry weight, respectively,
which were close to those reported for the wild S. naozhouense (13.95% and 41.79%) [19] and higher
than those of Saccharina japonica (8.70% and 20.00%) [22]. Furthermore, the protein content was
comparable to that recorded for some species of the same genus, i.e., S. henslowianum (11.52%) and
S. fusiforme (15.38%) [23,24].
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Table 1. Chemical composition of cultivated S. naozhouense (%, w/w on the dry basis) a.
Components
Ash
Protein
Lipid
Total carbohydrate
Total water-soluble carbohydrate
Water-soluble polysaccharide
Total dietary fiber
a
Values
35.18
11.20
1.06
47.73
29.74
21.01
4.83
Average of four analyses.
Interestingly, the total carbohydrate level (47.73%) was higher than that reported for S. fusiforme
(46.01%) and wild S. naozhouense (29.37%) [19,24]. Moreover, the main carbohydrates were
water-soluble polysaccharides (21.01%), yet the dietary fiber content (4.83%) was relatively lower. On
the other hand, the lipid content (1.06%) was relatively lower. This result was similar to that of wild
S. naozhouense (2.4%) and other edible brown algae, such as Saccharina japonica (0.2%) and
S. fusiforme (0.69%) [22,24].
2.2. Amino Acid Composition
The amino acid composition of proteins in cultivated S. naozhouense was illustrated (Table 2). The
contents of amino acids ranged from 0.54 to 13.21 g/100 g protein. The proteins of cultivated
S. naozhouense contained a high level of amino acids, especially essential amino acids (EEA),
e.g., leucine (6.52 g/100 g protein) and valine (4.64 g/100 g protein). Furthermore, all essential amino
acids, such as valine, methionine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine and
tryptophan, accounting for 47.22% of the total amino acids, were present in this seaweed. The ratio
value of EAA/NEAA and the essential amino acid index (EAAI) were 0.89 and 66.24, respectively.
According to FAO/WHO recommended standards of ideal protein [25], the protein of cultivated
S. naozhouense belongs to a high-quality protein. Furthermore, the protein quality is better than that of
S. fusiforme, because cysteine is lacking in S. fusiforme [24].
In addition, the aspartic (8.39 g/100 g protein) and glutamic acids (13.21 g/100 g protein),
non-essential amino acids (NEEA), were the most abundant amino acids and accounted for 28% of
total amino acids, which, together with glycine (4.38 g/100 g protein) and alanine (5.27 g/100 g
protein), were responsible for the special flavor and taste of cultivated S. naozhouense.
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Table 2. Amino acid composition of cultivated S. naozhouense (g/100 g protein) a.
Amino acids
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Methionine
Cysteine
Isoleucine
Leucine
a
Contents
8.39
3.93
3.21
13.21
3.30
4.38
5.27
4.64
2.41
0.54
4.02
6.52
Amino acids
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Tryptophan
Total
EAA
NEAA
EAA/NEAA
EAAI
Contents
2.95
4.38
1.07
3.66
4.20
0.89
76.97
36.35
40.62
0.89
66.24
Average of four analyses; EAA: essential amino acids, Threonine, Valine, Methionine, Isoleucine, Leucine,
Phenylalanine, Lysine, Histidine, Arginine, and Tryptophan; NEAA: non-essential amino acids; EAAI:
essential amino acid index.
2.3. Fatty Acid Composition
The fatty acid composition of cultivated S. naozhouense is presented (Table 3). This seaweed
contained high concentrations of saturated fatty acids (SAFA, 33.63% of total of fatty acid),
monounsaturated fatty acid (MUFA, 10.42% of total of fatty acid), and polyunsaturated fatty acid
(PUFA, 18.84% of total of fatty acid), even though it had a low level of lipid.
The main fatty acids in cultivated S. naozhouense were C14:0 (myristic acid), C16:0 (palmitic acid),
C18:1 (oleic acid) and C20:4 (arachidonic acid), which were also the most abundant fatty acids in
edible seaweed S. fusiforme [24]. However, C16:0 (palmitic acid), C18:0 (stearic acid) and C18:1
(oleic acid) were the most abundant fatty acids in wild S. naozhouense [19].
Table 3. Fatty acid composition of cultivated S. naozhouense (% of total of fatty acid) a.
Fatty acids
C6:0
C8:0
C12:0
C14:0
C15:0
C16:0
C16:1
C16:2ω6
C18:0
C18:1
C18:2trans
C18:2ω6cis
a
Methyl esters (%)
0.44
0.49
0.34
6.7
0.3
24.61
3.55
0.22
0.54
6.34
3.97
0.46
Fatty acids
C18:3ω3
C20:1
C20:3ω6
C20:4ω6
C20:5ω3
C22:0
SAFA
MUFA
PUFA
PUFAω6
PUFAω3
Ratioω6/ω3
Methyl esters (%)
0.25
0.53
2.95
9.61
1.38
0.21
33.63
10.42
18.84
13.24
1.63
8.12
Average of four analyses; SAFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA:
polyunsaturated fatty acids.
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Although our study revealed that cultivated S. naozhouense had higher total levels of PUFA than
MUFA, the eicosapentaenoic acid (EPA, C20:5ω3) and essential fatty acids, such as C18:2ω6cis
(linoleic acid), C18:3ω3 (linolenic acid, and C20:4ω6 (arachidonic acid), the most interesting and
important fatty acids in terms of nutrition, were present in this seaweed. Further, the ratio of ω6/ω3,
which the WHO currently recommends should not be higher than 10 in diet as a whole [26], was 8.12,
which indicated the cultivated S. naozhouense may be used as a sea vegetable or an ingredient to
reduce ω6/ω3 ratio in diet.
2.4. Mineral Contents
Different mineral elements (such as potassium, sodium, phosphorus, calcium, iron, zinc,
manganese, copper and cadmium) were analyzed by inductive coupled plasma-optical emission
spectroscopy (ICP-OES) and were summarized (Table 4). The cultivated S. naozhouense contained
significant amounts of essential minerals (e.g., potassium, sodium, calcium and phosphorus), like
S. fusiforme and wild S. naozhouense [19,24]. Potassium (4170 mg/100 g dry weight) was the most
abundant element in the seaweed, followed by sodium (3250 mg/100 g), phosphorus (120 mg/100 g)
and calcium (66.98 mg/100 g). The ratio of Na/K (0.77) was relatively lower, which was interesting
from the point of view of nutrition, because high Na/K ratio diets and the incidence of hypertension are
closely connected [27]. Consequently, the cultivated S. naozhouense may be useful for the regulation
of the Na/K ratio of diets.
On the other hand, cultivated S. naozhouense also contained trace elements, such as iron,
manganese, zinc, copper and cadmium. Iron was the most abundant trace element (147 mg/100 g),
followed by Zn (9.08 mg/100 g). The content levels of other trace elements (Table 4) were similar to
those recorded in the earliest reports on seaweeds [15,28]. Furthermore, the contents of some heavy
metal elements (As, Cd, Cu, Hg and Pb) in this seaweed were below the toxic limits allowed in some
countries [29]. Hence, cultivated S. naozhouense may be used as a food supplement to provide the
daily intake of some trace elements (e.g., iron, zinc) for adults, especially iron, since iron deficiency
would lead to anemia, when the demand for iron is high in growth, high menstrual loss and pregnancy [27].
Table 4. Mineral composition of cultivated S. naozhouense (mg/100 g) a.
Minerals
K
Na
P
Ca
Fe
Zn
Mn
Cu
Cd
Contents
4170
3250
120
66.98
147
9.08
5.84
0.36
0.17
a
Average of four analyses.
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2.5. Properties of Polysaccharide
The content of water-soluble polysaccharides from cultivated S. naozhouense was 21.01% (Table 1).
The IR spectrum of polysaccharides was recorded in a potassium bromide pellet using an IR
spectrophotometer. In the IR spectrum (Figure 1), it is being observed that a broad peak at 3415 cm−1
and a small peak at 2930 cm−1 are due to the stretching vibrations of O–H and C–H, respectively. The
bands at 1613 and 1415 cm−1 were attributed to carboxylate O–C–O asymmetric stretching and to
C–OH deformation vibrations, respectively. The absorption at 1039 cm−1 was assigned to C–O and
C–C stretching vibrations of the pyranose ring. The anomeric region of the fingerprint (950–750 cm−1)
exhibited three characteristic absorption bands in alginate polysaccharides (Figure 1, bands at 896, 821
and 777 cm−1, respectively). The band at 896 cm−1 is assigned to the β-anomeric C–H deformation
vibration of β-mannuronic acid residues. The band at 821 cm−1 seems to be characteristic of
mannuronic acid residues. The band at 777 cm−1 is assigned to gluluronic acid [30]. In addition, a
broad band at 1249 cm−1 indicated the presence of sulphated ester groups (S=O), which is a
characteristic component in fucoidan [31–33]. Therefore, the water-soluble polysaccharides from
cultivated S. naozhouense may be alginates and fucoidan, which should be further demonstrated by
more studies in the future.
Figure 1. FT-IR spectrum of water-soluble polysaccharides from cultivated S. naozhouense.
2.6. Cytotoxic and Antiviral Activities of the Polysaccharides
The polysaccharides exhibited lower cytotoxicity on Vero cells (CC50, MCC > 200 μg/mL). After
Vero cells had been treated by various polysaccharide dilutions for two days, cell morphology did not
have any visible alteration under a phase-contrast microscope, and cell layer also did not have any
destruction by MTT reduction assay.
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The polysaccharide showed strong antiviral activity against HSV-1 strain F at ≥12.5 μg/mL
(EC50 = 8.92 μg/mL). In order to compare antiviral potential of the polysaccharides, acyclovir (ACV)
was used as a positive control and conferred more than 75% cellular protection at 20 μg/mL, which
was in agreement with that the polysaccharides at 12.5 μg/mL (Table 5). Moreover, the selectivity
index (SI, CC50/EC50), which was often higher than 10 for a sample with antiviral activity [7], was
more than 22. It is clear that water-soluble polysaccharides from cultivated S. naozhouense possess
anti-HSV-1 activity in vitro.
Herpes simplex virus type 1 (HSV-1), a common human pathogen, is responsible for a broad range
of human infectious diseases. Though ACV is served as a drug to successfully treat the HSV
infections, ACV-resistant strains have been found in immune-compromised patients and drug toxicity
has also been reported [34,35]. Therefore, searching for new antiviral agents is urgently needed.
Natural bioactive compounds are the best resources for the development of new anti-HSV drugs due to
their greater efficiency with less toxicity. Sulfated polysaccharides from marine algae, including
sulfated mannans, galactans, agarans, fucoidans, sulfated rhamnogalactans, fucans and different types
of carrageenans, were showed to be active against some enveloped viruses, especially HSV and
HIV [21,36–39]. In this study, the sulfated water-soluble polysaccharides showed strong antiviral
activity against HSV-1 strain F in vitro within noncytotoxic concentration. Consequently, the sulfated
polysaccharides from marine algae are a good resource for searching novel therapeutic candidates for HSV.
Table 5. Effect on HSV-1strain F replication in Vero cells and cytotoxicity of polysaccharide
from cultivated S. naozhouense.
Concentration
(μg/mL)
Polysaccharide
CPE
50
25
12.5
6.25
3.12
1.56
0.78
–
–
–
+
++++
++++
++++
++++
EC50 (μg/mL)
8.92
CC50 (μg/mL)
>200
MCC (μg/mL)
>200
Concentration
Acyclovir (ACV)
100
(μg/mL)
CPE
Virus
contrast
++++
20
+
―++++‖: >75% cytopathic; ―+++‖: 50%–75% cytopathic; ―++‖: 25%–50% cytopathic; ―+‖: 0%–25% cytopathic; ―–‖: no
cytopathic effect induced by virus.
3. Experimental Section
3.1. Algal Material
The cultivated S. naozhouense was collected from Techeng Island, Guangdong province of China in
July 2011 and identified by Professor Weixin Li and Dr. Xie Enyi, Fisheries College, Guangdong
Ocean University, China. A voucher specimen (No. P110701) was deposited in the Key Laboratory of
Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese
Academy of Sciences, Guangzhou, China. The fresh seaweeds were washed in freshwater to remove
sediment, epifauna and epiphytes, and then dried in the air for 10 h, powdered and stored in plastic
bags at 4 °C until further experiment use.
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3.2. Chemical Composition
Total nitrogen was quantified by the Kjeldahl method, and then, the protein content was estimated
by multiplying the total nitrogen content by a nitrogen conversion factor of 6.25 [8]. Total ashes were
determined by incinerating seaweed samples in a digitally controlled furnace with temperature being
gradually increased to 550 °C and maintaining for 6 h, and then were quantified gravimetrically [40].
Total lipids, total water-soluble carbohydrates and total dietary fibers were determined by the Soxhlet
method, anthrone-sulfuric acid colorimetry and the gravimetric method, respectively [40]. Total
carbohydrate was determined by the difference method [24]. Polysaccharide content was estimated by
the phenol-sulfuric acid method, using glucose as a standard substance [41]. Amino acids were
determined by high-performance liquid chromatography (HPLC) according to the GB/T 5009.124-2003
standard method [42]. Fatty acid composition was determined by gas chromatography-mass
spectrometry (GC-MS) analysis of their methyl esters on a Varian Gas Chromatograph series 3800
fitted with a VF-5 MS fused silica capillary column (30 m ×0.25 mm, film thickness 0.25 μm, USA) [43].
Mineral analysis was made by inductive coupled plasma-optical emission spectroscopy (ICP-OES) [43].
3.3. Preparation of Polysaccharides
Approximate 20.0 g seaweed powder were accurately weighed and defatted in a Soxhlet apparatus
with petroleum ether (60–90 °C), then pretreated twice with 80% ethanol to remove some pigments,
monosaccharides, oligosaccharides and other small molecule materials. After the organic solvent was
volatilized, the pretreated seaweed powder was extracted twice with distilled water at 90 °C for 1.5 h
and filtered. The combined aqueous extracts were concentrated in a rotary evaporator to a certain
volume under reduced pressure at 50 °C, followed by treatment with Sevag Reagent to remove protein
and centrifugation at 5000 rpm for 20 min to obtain the supernatant. Then the supernatant was poured
into six volumes of 100% ethanol and was kept at 4 °C overnight. The precipitate containing crude
polysaccharides was collected by centrifugation, then washed with 70% ethanol, 100% ethanol, ethyl
ether and acetone, respectively, and finally, freeze-dried, weighed and kept in a vacuum dryer [44–46].
3.4. FT-IR Spectroscopy
Infrared spectra were recorded from a KBr pellet of the polysaccharide on a spectrometer FT-IR
Nicolet 6700.
3.5. Determination of Antiviral Activity of the Sulfated Polysaccharide
3.5.1. Cells and Virus
African green monkey kidney cells (Vero, ATCC CCL-81), provided by Wuhan Institute of
Virology, Chinese Academy of Sciences, were cultured in Dulbecco’s modified Eagle medium
(DMED, Invitrogen) supplemented with 10% FBS (Invitrogen), 0.22% sodium bicarbonate (Sigma)
and 50 mg/L gentamycin (Invitrogen). HSV-1 strain F (ATCC VR733), obtained from Hong Kong
University, was propagated in Vero cells and stored at −80 °C until use.
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3.5.2. Cytotoxicity Assay
Cytotoxicity of the polysaccharides on Vero cells was evaluated in vitro by MTT assay as described
by Mosmann [47]. Vero cells (1 × 104 cells/well) were seeded in 96-well plates and incubated at 37 °C
in 5% CO2 atmosphere for 24 h. Then, various dilutions (concentration from 3.12 to 200 μg/mL) of
polysaccharide were added to wells, with quadruplicate wells for each concentration, and further
incubated for 48 h; meanwhile, cells were examined daily under a phase-contrast microscope to
determine the minimum concentration of polysaccharide (MCC) that caused a microscopically
detectable alteration of cell morphology. Afterwards, MTT solution was added (final concentration
0.5 mg/mL) to each well. After 4 h of incubation at 37 °C, the supernatant was removed, and the
dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, then the optical density
(OD) was measured in a microplate reader at 570 nm. The cytotoxicity was expressed as 50%
cytotoxic concentration (CC50), which was the concentration of the test substances required to reduce
cell growth by 50%.
3.5.3. Antiviral Activity Assay
The antiviral activity of the polysaccharide was evaluated by cytopathic effect (CPE) inhibition
assay [48]. In general, Vero cells were seeded in 96-well plates at a density of 1 × 104 cells per well
and allowed to form a monolayer. The confluent cell monolayer was treated with serial two-fold
dilutions of polysaccharide and an equal volume of virus suspension (100TCID50) in quadruplicate in
96-well plates and then incubated at 37 °C in a 5% CO2 atmosphere and observed daily for CPE under
a light microscope. Meanwhile, acyclovir (ACV) was served as a positive control. The 50% effective
antiviral concentration (EC50), defined as the concentration that reduced CPE by 50% with respect to
the virus control, was calculated by MTT method.
4. Conclusions
The edible cultivated brown algae S. naozhouense was investigated for its potential nutritional value
and antiviral activity for the first time. It is characterized by a high level of proteins and a low level of
lipid, like S. fusiforme and Saccharina japonica used in human nutrition [18] and, therefore, can be
used for human consumption as an alternative source of essential amino acids and some
polyunsaturated acids, such as oleic, linoleic, linolenic and eicosapentaenoic acids, or as functional
ingredients to reduce calories and modify the texture of formulated foods. This seaweed is also rich in
some minerals, such as iron and zinc, and so may be used as a food supplement to supply these
minerals at low inclusion levels. Especially, the protein in cultivated S. naozhouense was better than
that in S. fusifome, because cystein was present in the former. In this regard, cultivated S. naozhouense
has higher nutritional value compared to S. fusiforme, which has been consumed as a longevity sea
vegetable in Chinese traditional diets and may be a better alternative resource of the Traditional
Chinese Medicine S. fusifome.
On the other hand, cultivated S. naozhouense has a high level of water-soluble polysaccharides,
which is very interesting, because pharmacological and biological activities of polysaccharides from
marine algae in therapeutic applications for humans are already well-known [49]. Furthermore, the
Mar. Drugs 2013, 11
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water-soluble polysaccharides contained sulfate groups and exhibited strong antiviral activity against
HSV-1 strain F in vitro with an EC50 of 8.92 μg/mL and a SI of >22. Hence, the polysaccharide from
cultivated S. naozhouense has a potential in antiviral new drugs. Further studies should be carried out
for the isolation, characterization and other biological screening of the polysaccharide.
In summary, the brown alga S. naozhouense may represent an interesting advance in the search for
novel functional applications in relevant industrial uses, including pharmaceuticals, nutraceuticals,
cosmeceuticals and functional foods.
Acknowledgments
This study was supported by the National Basic Research Program of China (973 Program,
Nos. 2010CB833800 and 2011CB915503), the National High Technology Research and Development
Program (863 Program, 2012AA092104) and the National Natural Science Foundation of China
(Nos. 30973679, 21172230, 31270402, and 21002110), Guangdong Province-CAS Joint Research
Program (2011B090300023).
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